High performance capillary gel-electrophoresis has proven to be a powerful technique in the separation of oligonucleotides and medium to high molecular weight polypeptides and proteins as discussed in U.S. Pat. Nos. 4,865,706, 4,865,707, and U.S. pat. app. Ser. No. 07/406,080 hereby incorporated by reference. This technique substantially out-performs more traditional methods of electrophoretic separations such as those conducted on flat plates or slabs.
One important consideration of related to electrophoresis in a capillary is the elimination of electroosmotic flow. A capillary is particularly sensitive to the effects of electroosmotic fluid flow which decreases the resolution of some electrophoretic separations.
In the past, researchers in the field have attempted to control electroosmotic flow and other surface zeta potential associated electrophoretic phenomena by coating the internal surfaces of the electrophoresis tube with various materials to eliminate the characteristic charges associated with those surfaces. For example, in U.S. Pat. No. 3,728,145, Hjerten discloses a method for coating the inner wall of a large bore tube with a neutral hydrophilic substance such as methyl cellulose or polyacrylamide to reduce electroosmosis in free-zone electrophoresis in open tubes. In a later patent, U.S. Pat. No. 4,860,201, Hjerten discloses a method for coating the inner wall of a narrow bore capillary with a monomolecular coating of polyacrylamide bonded to the capillary wall by means of a bifunctional reagent. These capillaries are also open tubes to be used for free-zone electrophoresis. In the background of the '201 patent, it is stated that coating the inner wall of the electrophoresis tube with a polymeric substance to reduce adsorption and electroosmosis suffers from the drawbacks that the coating material must be renewed frequently since it apparently flushes out of the capillary during use, and that relatively thick layers necessary for complete coating cause zone deformation in electrophoresis. This '201 patent thus teaches away from coating the wall of a capillary with a polymeric substance applied as an adsorbed layer, and discloses instead that for suppression of electroosmosis, a monomolecular layer of polyacrylamide should be covalently attached to the wall.
The small amount of work in the field of gel electrophoresis in capillaries by researchers other than the present inventors has generally resulted in columns which were not highly stable and could not be subjected to sufficiently high electric fields to achieve high efficiencies and high resolution separations.
The inventors have previously addressed the need for an improved coating to reduce the problem of electroosmotic flow in a gel-filled capillary in application Ser. No. 07/406,080. This application discloses a capillary column for high performance gel electrophoresis which includes a thin layer of coating material covalently bonded to the inner surface of the capillary wall, and a thin layer of a hydrophilic polymer adsorbed on the layer of coating material. It was found that the layer of hydrophilic polymer is highly stable, effectively reduces electroosmosis, stabilizes the column, and unexpectedly, enables operation of the capillary column in high electric fields (or more exactly, high power), resulting in high resolution separations.
The inventors have since found that high performance electrophoresis in a capillary can be even more powerful if the effects of electroosmosis in the capillary column can be further controlled, and if the capillary column itself is highly stable and capable of being reused numerous times.
Criteria such as those just described requires a capillary column in which the effects of electroosmosis can be made almost negligible, and which is highly stable so as to withstand repeated use in high electric fields and repeated washings.